Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form Tc-99m-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both Tc-99m and Re-188 using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)(2)S-2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)(2) groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with Tc-99m is achieved by direct reduction with Sn(II) tartarate to yield the Tc-99m-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with Re-188 is achieved by transchelation with Re-188 citrate in yields of greater than or equal to 90%. in this study, (PH2)(2)S-2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)(2)S-2-COOH BFCA conjugate was converted to the hydroxy methylene phosphine form and complexed with 99mTc to produce the (TcO2)-Tc-99m-P2S2-GlyGlyOEt conjugate 8 in RCPs of greater than or equal to 95%. This singular Tc-99m product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the (TcO2)-Tc-99m-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys(6)-LHRH conjugate and producing its corresponding Tc-99m complex in RCPs of greater than or equal to 88%. Finally, the P2S2-5-AVa-BBN[7-14] NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)(2) groups and subsequently labeled with Re-188 to yield a Re-188-labeled bombesin analogue with a RCP of greater than or equal to 90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)(2)S-2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (Tc-99m) and therapeutic (Re-188) radionuclides via the postconjugation approach in high yields.